Characterization and biological activities of a novel polysaccharide isolated from raspberry (Rubus idaeus L.) fruits

Article abstract of DOI:10.1016/j.carbpol.2015.06.068

Abstract A water-soluble polysaccharide namely RCP-II from raspberry fruits was obtained by complex enzyme method followed by successive purification using macroporous resin D4020 and Sephadex G-100 columns. RCP-II was an acidic heteropolysaccharide and the characteristic structure of polysaccharide was determined. The carbohydrate of RCP-II was composed with galacturonic acid, rhamnose, arabinose, xylose, glucose and galactose in a molar ratio of 1.00:0.55:1.19:0.52:0.44:1.90 and the average molecular weight was estimated to be 4013 Da, based on dextran standards. RCP-II presented high scavenging activity toward DPPH?, HO?, O₂^?- in a concentration-dependent manner. The determination of the inhibitory activity on protein glycation showed that in 14 days of incubation the inhibitory ability of RCP-II was more effective on the development of non-enzymatic glycation reaction at early phase than that at the following two phases.

Full text of DOI:10.1016/j.carbpol.2015.06.068

Carbohydrate Polymers 132 (2015) 180–186
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Characterization and biological activities of a novel polysaccharide
isolated from raspberry (Rubus idaeus L.) fruits
Zeyuan Yua, Lu Liua, Yaqin Xub, Libo Wangb,∗, Xin Tenga, Xingguo Lia, Jing Daia
a College of Horticulture, Northeast Agricultural University, Harbin 150030, China
b College of Science, Northeast Agricultural University, Harbin 150030, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A water-soluble polysaccharide namely RCP-II from raspberry fruits was obtained by complex enzyme
method followed by successive purification using macroporous resin D4020 and Sephadex G-100
columns. RCP-II was an acidic heteropolysaccharide and the characteristic structure of polysaccharide
was determined. The carbohydrate of RCP-II was composed with galacturonic acid, rhamnose, arabinose,
xylose, glucose and galactose in a molar ratio of 1.00:0.55:1.19:0.52:0.44:1.90 and the average molecular
weight was estimated to be 4013 Da, based on dextran standards. RCP-II presented high scavenging activ-
Received 7 February 2015
Received in revised form 17 June 2015
Accepted 20 June 2015
Available online 25 June 2015
Keywords:
Rubus idaeus L.
Polysaccharide
Purification
Antioxidant activity
Non-enzymatic glycation inhibition activity
•−
ity toward DPPH•, HO•, O2 in a concentration-dependent manner. The determination of the inhibitory
activity on protein glycation showed that in 14 days of incubation the inhibitory ability of RCP-II was
more effective on the development of non-enzymatic glycation reaction at early phase than that at the
following two phases.
© 2015 Elsevier Ltd. All rights reserved.
Chemical compounds studied in this article:
d-Glucose (PubChem CID: 5793)
d-Arabinose (PubChem CID: 66308)
d-Galacturonic acid (PubChem CID: 84740)
Trifluoroacetic acid (PubChem CID: 6422)
Ascorbic acid (PubChem CID: 54670067)
Carbazole (PubChem CID: 6854)
Girard-T (PubChem CID: 67156)
DPPH (PubChem CID: 2735032)
1. Introduction
of them are used in processed products such as juice, jam, wine
and milk shake (Vladisavljevic´, Vukosavljevic´, & Veljovic´, 2013).
In recent decades, it has been found that natural polysaccharides
extracted from edible fruits possess various important biolog-
ical activities, such as anti-cancer, antioxidant, immunological,
hypolipidemic, anti-inflammatory and hypoglycaemic activities
(Yang et al., 2006; Yang & Zhang, 2009; Cui et al., 2014; Dou et al.,
2015). Therefore, increasing attention is being placed on extraction
and characterization of new bioactive polysaccharides which may
be applied to functional foods and medicine.
As a member of the ‘berry family’, raspberry (Rubus idaeus L.) is
widely distributed in all temperate regions of Europe, North Amer-
ica and Asia (including the three provinces in Northeast China)
(Chen, Xin, Zhang, & Yuan, 2013). The fruits of raspberry are sub-
globose and bright red when they are matured, and the majority
Nowadays, raspberry gains most acceptance for its nutritive value,
as they contain numerous bioactive compounds such as phenolics,
ascorbic acids, minerals and anthocyanins (Kafkas, Özgen, Özog˘ul,
& Türemis, 2008; Bobinaite˙ , Visˇkelis, & Venskutonis, 2012).
In recent years, many biological activities of raspberry have
been found in many studies (Paredes-López, Cervantes-Ceja,
Vigna-Pérez, & Hernández-Pérez, 2010; Maksimovic´, Milivojevic´,
Poledica, Nikolic´, & Maksimovic´, 2013), however, there are few
reports on its polysaccharides, and the structure and function of
these polysaccharides have never been well characterized. Our pre-
vious study demonstrated raspberry was also an excellent source
of polysaccharides compared with other nutrients, and the yield of
polysaccharides was 4.12% (counted with the weight of wet fruits)
using complex enzyme method (Yu, Teng, Xu, & Li, 2014). In present
study, a novel low-molecular-weight polysaccharide (RCP-II) from
raspberry was isolated, and the structural characteristics were pre-
liminary determined. The antioxidant activity and non-enzymatic
∗
Corresponding author. Tel.: +86 451 55190732; fax: +86 451 55190243.
E-mail address: wanglibo99166@163.com (L. Wang).
http://dx.doi.org/10.1016/j.carbpol.2015.06.068
0144-8617/© 2015 Elsevier Ltd. All rights reserved.

Z. Yu et al. / Carbohydrate Polymers 132 (2015) 180–186
181
glycation inhibition activity of RCP-II were also investigated to
provide scientific evidence for further study.
1976), ninhydrin reaction, iodine–potassium iodide reac-
tion, Fehling reagent reaction (Li, Zhou, Cai, & Zhang, 1999),
carbazole–sulphuric acid reaction (Bitter & Muir, 1962), FeCl3
reaction. In addition, the content of esterified carboxyl was
determined by the titrimetric method (FCC, 1981).
2. Materials and methods
2.1. Materials
2.4. Characterization of RCP-II
Raspberry (R. idaeus L.) fruits, with cultivar name Autumn bliss,
were used in the experiment, which were obtained from the
horticulture station of Northeast Agricultural University (Harbin,
China) and stored at −18 ◦C until they were used. Before extrac-
tion, the frozen fruits were thawed at room temperature and then
homogenised using a JJ-2 homogenizer (Changzhou Guohua Elec-
tric Appliance Co., Ltd, Jiangsu Province, China).
D4020 Macroporous Resin was purchased from NanKai Uni-
versity Chemical Plant (Tianjin, China). Sephadex G-100 and
standard monosaccharides (d-glucose, d-galactose, d-rhamnose,
d-mannose, d-arabinose and d-fructose) were purchased from
Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). T-series dextran stan-
dards (T-3, T-5, T-10, T-40 and T-70) were obtained from Beijing
Baier Di Biotechnology Co., Ltd (Beijing, China). Ascorbic acid
(Vc) and bovine serum albumin (BSA) were produced by Tianjin
Regent Chemicals Ltd. (Tianjin, China) and Sino-American Biolog-
ical Engineering Co., Ltd. (Henan Province, China), respectively.
Pectinase (28.48 U/mg) and cellulase (13.83 U/mg) were obtained
from Shanghai Lanji Biotechnology Co., Ltd (Shanghai, China).
Papain (23.74 U/mg) was purchased from Beijing Obo Star Biotech-
nology Co., Ltd (Beijing, China). All other chemicals and reagents
were of analytical grade.
2.4.1. Determination of molecular weight
The molecular weight of RCP-II was measured by high per-
formance liquid chromatography (HPLC, LC-10AVP, Shimadzu
Corporation, Japan) with a refractive index detector (RID-10A).
The sample was dissolved in ultrapure water (1 mg/mL) and fil-
tered through a 0.45 ␮m membrane. Then 10 ␮L RCP-II solution
was applied to a gel-filtration chromatographic column of Waters
Ultra-hydroge 2000 (7.8 mm × 30 cm) and eluted with 0.1 mol/mL
NaNO3 at a flow rate of 1.0 mL/min. Standard dextrans (T-3, T-5, T-
10, T-40 and T-70) were separate passed through the column and
their retention time were plotted against the logarithms of their
respective molecular weights. The molecular weight of RCP-II at a
given retention time was calculated from the calibration equation
generated with the standard curve.
2.4.2. Analysis of monosaccharide composition
Gas chromatography (GC, GC-2010, Shimadzu Corporation,
Japan) equipped with
a RTX-1701 silica capillary column
(30.0 m × 0.25 mm × 0.25 ␮m) and a flame ionization detector (FID)
was used to analyze the monosaccharide components. Sample RCP-
II (30.0 mg) was hydrolyzed with 2.0 mL of trifluoroacetic acid
solution (TFA, 2.0 mol/mL) in a sealed glass tube at 100 ◦C for 2 h.
The solution was evaporated to dryness at 30 ◦C and dissolved
in 3 mL of methanol. This procedure was repeated until the TFA
and methanol were removed completely. Then the sample and all
standard sugars (d-galacturonic acid, d-glucuronic acid, d-xylose,
d-galactose, d-mannose, d-glucose, d-arabinose and d-rhamnose)
were acetylated following a published method (Ma et al., 2014) and
analyzed by GC under the temperature condition as follows: the
column temperature was initially set at 180 ◦C, increased to 220 ◦C
at the rate of 5 ◦C/min and was held at 220 ◦C for 5 min, then ele-
vated to 280 ◦C at 10 ◦C/min and finally maintained for 20 min. The
heater temperatures of detector and injector were 280 ◦C. Nitro-
gen was used as the carrier gas and maintained at 1.0 mL/min. The
content was calculated by peak area internal standard with inositol
taken as the internal standard.
2.2. Extraction and purification of polysaccharides
Polysaccharides from raspberry were extracted according to our
previous study (Yu et al., 2014). Homogenised raspberry fruit was
mixed with complex enzymes (pectinase, cellulase and papain;
2.5:1.7:2.1) at 2.6% concentration, then citric acid–disodium hydro-
gen phosphate buffer (pH = 4.0) was added at certain liquid to solid
ratios (10:1 mL/g). The enzymolysis reactions were carried out in a
shaking bath (55 ◦C) for 2.6 h. After centrifugation at 5000 rpm for
15 min, the supernatants were combined, concentrated by rotary
evaporator to a proper volume, precipitated by the addition of
ethanol to a final concentration of 85% (v/v) and then kept overnight
at 4 ◦C. The precipitates collected by vacuum filtration through a
0.45 ␮m microporous membrane (Shanghai Wanzi Shiye Co., Ltd,
Shanghai, China) were dissolved in distilled water, concentrated
and lyophilized, and then extracted polysaccharides were obtained.
The extracted polysaccharide solution (3 mg/mL) was injected
to the macroporous resin (D4020) column (2.0 cm × 30 cm) and
eluted with deionized water at a flow rate of 1.2 mL/min. The
eluate (5 mL/tube) was collected automatically and monitored by
phenol-sulfuric acid method at 490 nm (Masuko et al., 2005). One
major polysaccharide peak namely RCP was obtained, and then
lyophilized. The fraction was further purified by Sephadex G-100
column (2.6 cm × 60 cm) eluted with distilled water at a flow rate
of 0.3 mL/min (1 mL/tube) to yield peaks RCP-I and RCP-II using the
method mentioned above. The main fraction RCP-II was applied in
the subsequent studies.
2.4.3. UV and IR spectroscopy
UV spectrum of RCP-II was recorded with a double beam UV
spectrophotometer (TU-1901, Beijing Purkinje General Instrument
Co., Ltd, Beijing, China) in the wavelength range of 600–190 nm−1
.
IR spectrum of the RCP-II was determined by a FT-IR spectropho-
tometer (FTS135, BID-BAD Co., USA) over the wave number range
from 4000 to 500 cm−1 with KBr pellets.
2.4.4. NMR spectroscopy
20.0 mg RCP-II was dissolved in 2.0 mL D2O (99.9%) and trans-
ferred into a 5 mm NMR-tube. NMR spectra (1H NMR, 13C NMR)
were recorded by AVANCEIII NMR spectrometer (Bruker Corpora-
tion, Switzerland).
2.3. Physical and chemical properties analysis
2.5. Antioxidant activities of RCP-II
The morphology and color were observed directly. Solubility of
RCP-II in water and ordinary organic solvents (acetone, chloroform
and ethanol) were investigated.
Chemical properties of RCP-II were determined using the fol-
lowing methods: Coomassie brilliant blue reaction (Bradford,
2.5.1. Hydroxyl radical scavenging activity
The hydroxyl radical scavenging activity was measured by
Fenton reaction described by Zhong et al. (2013). Different
concentrations (0.2, 0.4, 0.6, 0.8, 1.0 mg/mL) of RCP-II solution
(2.0 mL) were incubated with a reaction mixture containing FeSO4

182
Z. Yu et al. / Carbohydrate Polymers 132 (2015) 180–186
(8.0 mmol/L, 2.0 mL), salicylic acid–ethanol solution (8.0 mmol/L,
2.0 mL) and H2O2 (8.8 mmol/L, 2.0 mL) at 37 ◦C for 30 min. The
absorbance of the solution was read at 510 nm by TU-1901
spectrophotometer (Beijing Purkinje General Instrument Co., Ltd,
Beijing, China). Vc was used as the positive control and deionized
water was used as the blank. The antioxidant activity on hydroxyl
radical was calculated with following formula:
against the blank of phosphate buffer. The inhibition rate (IR) was
calculated as follows:
ꢀ
ꢁ
A − Ac − Ad
Aa − Ab
IR % = 1 −
× 100
(4)
( )
where A is the absorbance of complete glycated system; A˛ is
the absorbance of glycated material without samples; Ab is the
absorbance of glycated material without samples and glucose; Ac
and Ad are the absorbance of reaction mixture without BSA and
glucose, respectively.
A0 − A1
Scavenging activity % =
( )
× 100
(1)
A0
where A0 is the absorbance of deionized water and A1 is the
absorbance of sample.
2.6.3. Analyses of dicarbonyl compounds
Dicarbonyl compounds were measured by Girard-T assay using
spectrophotometric analyses (Zhang et al., 2011). Briefly, the gly-
cated material (0.4 mL) and Girard-T stock solution (500 mmol/L,
0.2 mL) in sodium formate (500 mmol/L, 3.4 mL, pH = 2.9) were
incubated at room temperature for 1 h. The absorbance was tested
at 294 nm against a blank of sodium formate. Glyoxal was used as
a standard in the drawing process of the calibration curve which
was treated in a similar manner. IR was calculated as formula (4).
2.5.2. Superoxide anion radical scavenging activity
The superoxide anion radical scavenging activity was assayed by
the method of Li and Shah (2014) with some modifications. Briefly,
5.0 mL of Tris–HCl buffer (50.0 mmol/L, pH = 8.2) and 1.0 mL of RCP-
II sample (0.2, 0.4, 0.6, 0.8, 1.0 mg/mL) were mixed and stored for
20 min at 25 ◦C, then 0.3 mL of pyrogallol solution (3.0 mmol/L)
was injected into the mixture. The absorbance was measured at
320 nm every 60 s for 5 min. Vc was used as the positive control.
The superoxide anion radical scavenging activity was calculated by
the following equation:
2.6.4. Analyses of AGEs
ꢀA0 − ꢀA1
The glycated solution (0.5 mL) was diluted to 10 mL with
sodium buffer (200 mmol/L, pH = 7.4). The fluorescence was deter-
mined at 370 nm of excitation wavelength and 440 nm of emission
wavelength with LS45 spectrofluorometer (PerkinElmer Co., Ltd,
Waltham, MA, USA). IR was calculated according to the following
formula:
Scavenging activity % =
( )
× 100
(2)
ꢀA0
where ꢀA0 is the difference of absorbance values per 60 s for
different concentrations of samples and ꢀA1 is the difference of
absorbance values per 60 s for water without samples.
ꢀ
ꢁ
2.5.3. DPPH radical scavenging activity
F − Fc − Fd
F˛ − Fb
IR % = 1 −
× 100
(5)
( )
DPPH radical scavenging activity was measured according to
the modified method of Ye and Huang (2012). 2.0 mL of DPPH
solution (0.3 mmol/L DPPH in methanol) was mixed with RCP-II
solution (0.2, 0.4, 0.6, 0.8, 1.0 mg/mL). After the mixtures were
shaken and incubated at 25 ◦C for 30 min in the dark, the sample
absorbance values were measured at 517 nm. Vc was used as the
positive control at the same concentration. The scavenging activity
was calculated by the following equation:
where F is the fluorescence of complete glycated material. F˛, Fb, Fc
and Fd are the fluorescence of reaction mixture without samples,
samples and glucose, BSA, glucose, respectively.
2.7. Statistical analysis
A0 − A1
All presented data were expressed as mean standard deviation
of three determinations, followed by analysis of statistical signif-
icance using SPSS version 12.0 software. Results for p < 0.05 were
considered to be statistically significant.
Scavenging activity % =
( )
× 100
(3)
A0
where A0 is the absorbance of samples and A1 is the absorbance of
the DPPH solution without samples.
2.6. Non-enzymatic glycation inhibition activity
3. Results and discussion
2.6.1. Glycation of BSA
3.1. Extraction and purification
The BSA/glucose model was employed to evaluate the ability of
polysaccharide antiglycation (Wu, Hsieh, Wang, & Chen, 2009). The
total 20 mL of reaction mixture contained BSA (20 mg/mL, 10 mL),
glucose (500 mmol/L, 5 mL), and samples with different concentra-
tions dissolved in phosphate buffer (200 mmol/L, pH = 7.4, 5 mL).
The negative control only contained BSA or glucose under the same
conditions. All the mixtures were incubated in the dark at 37 ◦C and
2.0 mL of the glycated materials were taken from the whole system
for the following experiments in 0, 2, 4, 6, 8, 10, 12, 14th days.
The extracted polysaccharides were obtained from raspberry by
complex enzyme extraction, ethanol precipitation, and lyophiliza-
tion. The yield of 4.09% (counted with the weight of wet fruits) was
higher than the yield of 3.44% by hot water extraction (Wang & Xu,
2007). After purification by macroporous resin D4020, the polysac-
charide of RCP was obtained and further purified by Sephadex
G-100 column to yield RCP-I and RCP-II. The yield of RCP, RCP-I and
RCP-II were 2.26%, 0.08% and 0.41%, respectively. Because of the low
yield of RCP-I, only RCP-II was collected and used for further study.
The total sugar content of RCP-II was determined to be 92.76% by
phenol–sulfuric acid method and the content of uronic acid was
23.6% determined by carbazole–sulphuric acid method. According
to the result of chemical titration, the content of esterified carboxyl
was determined as 37.5%. In addition, RCP-II had no absorption peak
at 260 nm, 280 nm and 520 nm in UV spectrum, which indicated
that nucleic acid, protein and pigment were removed basically.
2.6.2. Analyses of Amadori products
The Amadori products were determined by the method of nitro
blue tetrazolium (NBT) reductive assay (Zhang, Wang, & Dong,
2011). The glycated material (0.5 mL) and NBT reagent (0.3 mmol/L,
2.0 mL) were added to the sodium carbonate buffer (100 mmol/L,
2.5 mL, pH = 10.35). Then the mixture was incubated at room tem-
perature for 20 min, and absorbance of sample was read at 530 nm

Z. Yu et al. / Carbohydrate Polymers 132 (2015) 180–186
183
Fig. 3. IR spectrum of RCP-II.
3.4. IR spectra analysis of RCP-II
The FT-IR spectra of RCP-II ranging from 4000 to 400 cm−1 were
presented in Fig. 3. The intense broad band around 3430 cm−1 and
the weak absorption band at about 2932 cm−1 were due to O H and
Fig. 1. HPLC elution profile of RCP-II.
C
H stretching vibration, respectively (Jiang et al., 2013). Further-
3.2. Physicochemical property analysis
more, the absorption peaks of 1732 and 1621 cm−1 were attributed
to the absorption of ester carbonyl ( COOR) groups and carboxylate
ion stretching band ( COO ), respectively (Zha et al., 2014), which
was consistent with the result of carbazole–sulphuric acid reaction.
Freeze-dried RCP-II appeared as odorless white powder. RCP-II
was soluble in water and practically insoluble in acetone, chlo-
roform, and ethanol. It had negative response to ninhydrin and
coomassie brilliant blue reactions, indicating no amino acids or
protein remained in the powder. The absence of starch, phenols
and reducing sugar in RCP-II was confirmed by iodine–potassium
iodide, FeCl3 and Fehling reagent reactions, respectively.
The peak at 1234 cm−1 was related to non-symmetrical C
O C
stretching vibration. Three absorption peaks within the range of
1100–1010 cm−1 suggested the possible presence of pyranoid ring
in RCP-II (Li & Shah, 2014). In addition, the characteristic absorption
peaks around 818 cm−1 and 922 cm−1 were ascribed to ␣-glycoside
bond and ␤-glycoside bond, respectively (Li et al., 2014).
3.3. Molecular weight and monosaccharide composition of RCP-II
3.5. NMR spectroscopy analysis
The elution peak of RCP-II was single, narrow and sharp in HPLC,
with the feature of homogeneity (Fig. 1). According to the regres-
sion equation of the standard curve made by different dextran
standards (y = −0.475x + 11.517, R2 = 0.9924), the molecular weight
of the purified RCP-II was calculated to be 4013 Da with retention
time of 16.660 min.
The GC profile of RCP-II with acid hydrolysis and acetylation
(Fig. 2) was obtained based on the retention time. According to the
monosaccharide standards, RCP-II was composed of d-galacturonic
acid, d-rhamnose, d-arabinose, d-xylose, d-glucose and d-galactose
in a molar ratio of 1.00:0.55:1.19:0.52:0.44:1.90. These results indi-
cated that RCP-II was an acidic heteropolysaccharide. By comparing
with the reported data, monosaccharide composition of RCP-II was
different from the polysaccharides in other berries, such as bilberry,
black currant and wild raspberry (Hilz, Bakx, Schols, & Voragen,
2005; Fu et al., 2015).
The 1H and 13C NMR spectra of RCP-II were shown in Fig. 4a
and b. The 1H NMR spectrum was crowded in a narrow region
ranging from 3 to 5 ppm which is typical signal of polysaccharides
(Nep & Conway, 2010). As could be seen in Fig. 4a, the chemical
shifts of C1 protons were higher or lower than 5.0 ppm, indicat-
ing the existence of both ␣- and ␤-configuration in RCP-II. The
signals in ı95–101 ppm corresponded to ␣-anomeric carbons and
the signals in ı101–105 ppm corresponded to ␤-anomeric carbons
in 13C NMR spectrum, which was consistent with the 1H NMR
spectrum (Huang, Li, Li, Wang, 2011). The signal at ı5.20 ppm
in 1H NMR spectrum was due to H1 resonance of ␣-galactose
residues and the signal at ı98.02 ppm in 13C NMR spectra sup-
ported this deduction. The signal at ı4.11 ppm in 1H NMR spectrum
and ı101.43 ppm in 13C NMR spectrum suggested the polysac-
charide contained ␤-arabinose residues (Yu & Yang, 1999). The
Fig. 2. GC profile of RCP-II with acid hydrolysis and acetylation.

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Z. Yu et al. / Carbohydrate Polymers 132 (2015) 180–186
signals at 16–18 ppm were assigned to the C-6 of the rhamnose
residues (Wang, Liu, & Fang, 2005). These results were also con-
firmed by GC analysis. Signals at 2.08 ppm (1H NMR), correlating
with carbon atoms at 19.89 ppm (13C NMR) were due to the O-acetyl
groups of galacturonic acid residues (Tamaki, Konishi, Fukuta, &
Tako, 2008). Signals at ı179.59 and ı176.07 ppm (13C NMR) corre-
sponded to the C-6 of unesterified and esterified galacturonic acid
units (Li, Ai, & Yang, 2013), which was consistent with the results
of carbazole–sulphuric acid reaction and IR data.
3.6. Biological activities of RCP-II
3.6.1. Assay of antioxidant activity
Hydroxyl radical scavenging activities of RCP-II and Vc were
determined and shown in Fig. 5a. The scavenging activity of RCP-II
directly increased with the promoting concentration and had a sig-
nificant difference (p < 0.01) within the range (0.2–1.0 mg/mL). The
scavenging ability of RCP-II was lower than that of Vc at every con-
centration point. The IC50 values of RCP-II and Vc were 0.96 mg/mL
and 0.63 mg/mL, respectively.
As could be seen in Fig. 5b, both RCP-II and Vc showed
obvious scavenging activity on superoxide anion radicals. The scav-
enging activity of RCP-II was concentration-dependent, but Vc
exhibited high scavenging activity (87.1 1.13%) at low concen-
tration (0.2 mg/mL). The maximum scavenging rate of RCP-II was
77.59 1.30% at 1.0 mg/mL, whereas that of Vc was 97.52 0.92%
at the same concentration. The IC50 value of RCP-II was 0.49 mg/mL
after the calculation.
As shown in Fig. 5c, the DPPH scavenging activity of RCP-II was
increased rapidly within the test dosage range. At the concentra-
tion of 1.0 mg/mL, RCP-II showed scavenging rate of 40.01 1.09%,
which is close to that of Vc (42.14 1.27%). This ability was lower
than that of hydroxyl radicals (52.77 0.53%) and superoxide anion
radicals (77.59 1.30%). On the basis of the results above, RCP-II
showed the strongest scavenging activity against the superoxide
anion radicals.
Fig. 4. (a) 1H NMR spectrum of RCP-II, (b) 13C NMR spectrum of RCP-II.
Published data indicate that some fruit polysaccharides also
have strong antioxidant activities and could be developed into
Fig. 5. Antioxidant effects of RCP-II: (a) hydroxyl radical scavenging activity, (b) superoxide anion radical scavenging activity, (c) DPPH radical scavenging activity.

Z. Yu et al. / Carbohydrate Polymers 132 (2015) 180–186
185
Fig. 6. Inhibitory effects of RCP-II on protein nonenzymatic glycation: (a) Inhibit Amadori product formation, (b) inhibit dicarbonyl compound formation, (c) inhibit AGEs
formation.
health food (Yang & Zhang, 2009). Compared to the antioxidant
activities of polysaccharides (TYAP-1, TYAP-2 and TYAP-3) from
thinned-young apple, RCP-II had more prominent scavenging activ-
ity on DPPH•, HO•, O2 (Dou et al., 2015). But at the same tested
inhibition increased significantly (p < 0.01) from 0.05 to 0.40 mg/mL
at the culture time of 14 day and the maximal inhibitory rate was
38.04 0.54%.
•−
Contrasting the inhibition of non-enzymatic glycation of pro-
teins in these three phases, the IC50 values of RCP-II were 0.26, 1.63
and 0.77 mg/mL, respectively. These results indicated that early
phase was more effective than the following two phase. It was
reported that NBT reduction could also be caused by superoxide
radical anion that formed by glucose autoxidation (Wolff & Dean,
1987). Based on antioxidant experimental results, RCP-II could be
used as effective superoxide anion radical scavengers according to
its strong scavenging ability. Thus, these results showed that the
inhibitory effects of RCP-II on non-enzymatic protein glycation may
associate with its antioxidative activity.
concentrations, the scavenging effects of WFPs from wolfberry
fruits against these three radicals were slightly higher than RCP-II
(He, Yang, Jiao, Tian, & Zhao, 2012). The activity differential between
the polysaccharides may be due to their different structure, such
as monosaccharide composition, molecular weight and the con-
tent of uronic acid. In the future, RCP-II might be expected to play
a promising role as dietary free radical scavengers for oxidative
damage prevention.
3.6.2. Non-enzymatic glycation inhibition activity
Schiff base and Amadori products are formed during the early
stages of non-enzymatic glycation of proteins. Amadori products
can reduce NBT in alkaline solution to yield a coloured reactant
with a maximum absorption at 530 nm (Baker, Zyzak, Thorpe, &
Baynes, 1994). As shown in Fig. 6a, the inhibitory activities of dif-
ferent dosages of RCP-II increased with the culture proceeding from
0 to 12 days and then decreased slowly. RCP-II showed the high-
est inhibitory ability of 54.68 0.49% under the concentration of
0.40 mg/mL at the culture time of 12 day. Moreover, the inhibitory
rate had a significant difference (p < 0.01) in the tested concentra-
tions (0.05–0.40 mg/mL) of RCP-II.
Many studies had showed that dicarbonyl compounds can yield
stable AGEs by inducing cross-linking of protein (Chen, Liu, Liu,
& Zhao, 2012). According to our research (Fig. 6b), the inhibitory
rate of different dosage increased gradually from 0 day to 14 days
of incubation. The peak inhibition of 27.84 1.26% was observed
in the group treated with 0.4 mg/mL RCP-II at the culture time of
14 day.
4. Conclusion
A
novel water-soluble polysaccharide named RCP-II was
isolated from raspberry fruits, which was an acidic heteropolysac-
charide mainly composed of galacturonic acid, rhamnose,
arabinose, xylose, glucose and galactose in a molar ratio of
1.00:0.55:1.19:0.52:0.44:1.90. RCP-II was a low-molecular-weight
polysaccharide and the average molecular weight was 4013 Da. The
characteristic absorptive peaks of polysaccharide structure were
determined by IR and the presence of ␣-galactose and ␤-arabinose
residues were confirmed by NMR analysis. RCP-II was proved to
have certain antioxidant capability and non-enzymatic glycation
inhibition activity. All of these findings provide a scientific basis for
the further use of polysaccharides from raspberry fruits.
Acknowledgments
Fluorescence intensity can also be used to measure the degree
of non-enzymatic glycation system and the inhibition activity of
samples (Zhang et al., 2011). The result was showed in Fig. 6c,
the inhibition increased slowly during the first 4 days and then
increased faster significantly and became flat after 12 days. The
This work was financially supported by the Scientific and Tech-
nological Research Project Foundation of Heilongjiang Provincial
Education Department (12531023) and Natural Science Foundation
of Heilongjiang Province of China (C2015004).

186
Z. Yu et al. / Carbohydrate Polymers 132 (2015) 180–186
Masuko, T., Minami, A., Iwasaki, N., Majima, T., Nishimura, S. I., & Lee, Y. C. (2005).
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